The long term goal of this project is to understand the molecular basis of induction of the E. coli SOS response and the resulting mutagenesis. First, we will identify additional genes in the SOS regulon by cloning their regulatory regions upstream from a galK reporter gene in low and high copy number plasmids. LexA binding sites will be located by footprinting and genetic experiments. Since recent experiments have suggested that LexA protein may recognize a second type of operator, the interaction of LexA protein with such operators will be studied using methods previously established for the recA operator. Second, with the coming availability of a crystal structure for RecA protein, we will attempt to define domains which specify RecA functions in the SOS response by using information from recA mutants. This project will involve production of a database of sequence and functional information of already existing mutants. To add to this database, we will measure the cleavage of LexA and UmuD proteins in vivo in uninduced and UV-induced recA80, recA432 and recA433 mutants and analyze the cleavage properties of the purified mutant RecA proteins. Third, we will continue analysis of the signalling mechanism which induces the SOS response by activating RecA protein. We will map fragments of chromosomal DNA which prevent induction of the SOS response when these fragments are present on high copy number plasmids. We will identify the functions of the genes which are present as regulators of the SOS induction pathway. Finally, the relationship between induction of the SOS regulon and enhanced excision of pyrimidine dimers from the transcribed DNA strand will be investigated.